SUMMARY Pseudomonas aeruginosa is a ubiquitous bacterium in nature and an important opportunistic human pathogen that can cause severe infections in patients with burns, severe wounds, pneumonia, and undergoing nosocomial procedures, especially those requiring intubation or catheterization. Clearing P. aeruginosa (Pa) has become progressively more difficult with growing antibiotic resistance. Of particular concern, Pa is the major cause of pulmonary infections in cystic fibrosis (CF) patients with over 70% of CF patients being colonized by adulthood. These infections decrease lung function that often reduce patient quality of life and cause early death. While there have been of Pa vaccines in development, none have been licensed. Like many Gram-negative pathogens, Pseudomonas spp. possess a type III secretion system (T3SS), a virulence factor that allows avoidance of host innate immunity and is required for the onset of infection. Structurally resembling a molecular syringe with an external needle, the T3SS apparatus (T3SA) provides an energized conduit from the bacterial cytoplasm into the host cell. A needle tip protein and the first of two translocator proteins localize to the distal end of the T3SA needle to mediate host cell contact. These proteins, PcrV and PopB, respectively, are required for Pseudomonas pathogenesis and are 95-98% conserved among Pseudomonas spp. The equivalent conserved Shigella T3SA tip and translocator proteins, IpaD and IpaB, respectively, were fused to give DBF, which we have shown as a novel subunit vaccine antigen protecting mice against challenges by homologous and heterologous Shigella spp. Similarly, highly conserved T3SA tip and translocator fusions have protected mice against Bordetella spp., Salmonella enterica and Burkholderia spp. In a preliminary mouse experiment, PaF, the PcrV/PopB fusion, was admixed with a mucosal adjuvant and delivered intranasally. In this initial preliminary experiment, 100% protection (with 44% sterilizing immunity) was seen for the PaF vaccinated mice. However, 60% of the PBS control mice also survived (although with no sterilizing immunity). Nevertheless, in this high risk, high reward R21, we will optimize the vaccine in the mouse model and to extend the findings to the accepted rat model of cystic fibrosis. Thus, because the PaF+dmLT vaccine appears to protect mice against Pa pulmonary challenge with 44% sterilizing immunity, PaF+dmLT will elicit a humoral and cell-mediated immune response that protects against most Pa strains, including those that cause disease in CF patients. The two specific aims of this investigation are: 1) Assess the respective humoral and cell-mediated immune responses elicited by PaF+dmLT and 2) Determine the protective efficacy of PaF+dmLT against Pa.